Key area 2.7: Genetic control of metabolism

1. Key area 2.7: Genetic control of metabolism

2. Selection and isolation of micro-organisms • Wild strains of micro-organisms can selected to be of use in industrial processes. • They are then cultured in an enriched nutrient medium, and pure strains are isolated.

3. Strain improvement • These strains may still need to be improved, for example: • To improve genetic stability • To improve their ability to grow on low cost nutrients • To produce large quantities of target compound • To allow easy harvesting of target compound after fermentation is complete • These improvements can be brought about by mutagenesis, breeding programmes or recombinant DNA technology.

4. Mutagenesis Mutagenesis is the creation of mutations. • What is a mutation? In nature mutations are rare and spontaneous and are the cause of variation. The rate of mutagenesis can be increased by exposing organisms to mutagenic agents. • List all the mutagenic agents you are aware of.

5. Mutagenesis • Sometimes a mutant strain produced shows improved characteristics such as increased yield. • However, mutant strains are often genetically unstable, and so may undergo reversed mutation (i.e. revert to the less useful wild-type state). • Therefore, before the improved strain is used in the industrial fermenter, it must be monitored to ensure that it is still in its mutated state.

6. Breeding programmes • Bacteria reproduce asexually. How many parents are involved and do the offspring show variation? • New strains of some bacterial species can arise due to horizontal transfer of genetic material. • Here plasmids or pieces of chromosomal DNA are transferred from one strain to another. • New strains are also produced by bacteria taking up and incorporating DNA fragments from their surroundings.

8. Recombinant DNA technology • With this technique scientists can transfer gene sequences from one organism to another and even from one species to another. • Plant or animal gene sequences can be transferred to microorganisms to produce plant or animal protein. • As a safety mechanism genes are often introduced that prevent the survival of the microorganism in an external environment.

9. By introducing genes to a micro-organism (bacterium or yeast) the following improvements can be made: • Amplify specific metabolic steps in a pathway to increase yield of target compound • Cause cell to secrete product into surrounding medium for ease of harvesting • Prevents microbe from surviving in the external environment to reduce chances of an outbreak.

10. Artificial transformation of a bacterium by recombinant DNA technology • Go back to your N5 notes and briefly describe the process of genetic engineering • The newly transformed (host) cell now contains a combination of its own DNA and that from another source joined together – it is said to contain recombinant DNA.

12. Restriction endonucleases • A restriction endonuclease is an enzyme which is used: • to cut up DNA (containing the required gene from the donor organism) into fragments • to cut open the bacterial plasmids that are to receive it. • Each restriction endonuclease recognises a specific short sequence of DNA bases called a restriction site.

13. Restriction endonucleases • The target sequence (4-8 nucleotides in length) is found on both DNA strands but running ion opposite directions. • The enzyme cuts both DNA strands and may produce blunt or sticky ends.

14. DNA ligase • DNA ligase is an enzyme which seals sticky ends (and blunt ends together). • It is used to seal a DNA fragment into a bacterial plasmid to form a recombinant plasmid containing recombinant DNA.

15. The recombinant plasmid is called a vector because it carries the DNA from the donor organism into the host cell. To be an effective vector the plasmid must contain restriction sites and marker genes, in addition to genes for self-replication and regulatory sequences to allow the control of gene expression. restriction site Vector (carrier) (marker gene)

16. Restriction site • The same restriction endonucleases must be able to cut open the restriction site as used to cut the DNA containing the required gene – the sticky ends must be complementary.

17. Marker gene • This gene enables scientists to determine whether a host cell has successfully taken up the plasmid vector. • For example, a plasmid could contain the marker gene for resistance to the antibiotic ampicillin. Therefore when cultured in a medium containing ampicillin, any host cells that have failed to take up a recombinant plasmid die since they lack the resistance gene.

18. Limitations of prokaryotes • Eukaryotic and prokaryotic DNA are different, and this can cause problems when trying to insert a gene from eukaryotes to prokaryotes. • For instance, eukaryotes contain introns interspersed among exons. • https://www.youtube.com/watch?v=MIfDx417SDs&list=PLb0WW0k29aHpIR44YLwrQol_cgisaDw-B

19. Ethics Common vs rare disorders • Pharmaceutical companies invest a lot of money in research and development of new products but must also make a profit. • They may have a tendency to finance the development of a drug for a common condition rather than a rare one (or a disorder that in confined to a developing country) Why?

20. Ethics Patents • A GMO (genetically modified organism) can be protected by a patent – a government certificate that grants the inventor the sole right to make, use and sell the invention for a limited period of time, even though it is not a novel invention and is using naturally occurring organisms. There is much controversy surrounding this.

21. Regulations surrounding the product: It must pose no danger to staff who manufacture it It is safe for consumers It is pure and uncontaminated It is fit for purpose (does what it says on the tin!) Regulations surrounding the manufacturing process: Maintains specific standards to ensure the product is pure. Uses safe, well designed facilities. Risk assessment

23. How much do you know? Higher Biology for CfE P207 Testing Your Knowledge 1 Q1-3 Higher Biology for CfEP216 Testing Your Knowledge 2 Q1-5 Applying Knowledge and Skills P219 Q5-8

24. Key words • Ethical issue • Genetic vector • Hazard • Mutagenesis • Recombinant DNA technology • Restriction endonuclease • Risk

25. Extended Responses Give an account of the different culture conditions required for the growth of microorganisms. Give an account of the phases of growth of microorganisms cultured in a ferementer.